It is known to produce recombinant proteins in gram negative host bacteria, for example E.coli, Pseudomonas, Vibrio spp. and Methylophilus methylotrophus.
One difficulty that can be encountered in this approach is that E.coli, and other such gram negative host bacteria useful for the expression of heterologous proteins are able to produce bacterial toxins such as endotoxins, which are integral lipo-polysaccharide (LPS) components of the bacterial cell wall, and which can be highly pyrogenic and can cause febrile reactions in animals.
It is desirable to reduce contaminating endotoxins in biological preparations when they are encountered.
There are known bioassays to detect bacterial endotoxin contamination of biological materials: the test used is the Chromagenic peptide endpoint method and this test has been adopted by the European Pharmacopoeia Commission and is described in 1999 European Pharmacopoeia--Supplement 1999, and can be used when needed for the purposes of the present invention.
Certain methods intended to remove or reduce levels of contaminating endotoxin from biological materials e.g. proteins are already known in the art.
Anion exchange chromatography for endotoxin removal is discussed by K. Khandake in an article entitled "Effective Removal of Negatively Charged Interfering Moleculed from Proteins" (U.S. Bulletin 2204, a technical bulletin from BioRad lnc., N.J., U.S.A.). Such chromatography is also discussed in "Why Anion Exchange Works So Well for Endotoxin Removal . . . Sometimes", (P. Gagnon, in Summer 1998 issue of "Validated Biosystems Quarterly Resource Guide for Downstream Processing, Validated Biosystems Inc., Tucson, Ariz., U.S.A.) K. C. Hou and R. Zaniewski, in Biotechnology and Applied Biochemistry 12, 315-324, 1990, describe endotoxin separation from albumin and gamma-globulin solutions using anion-exchange polymeric matrices carrying DEAE or QAE functional groups.
T. E. Karplus et al., in Journal of Immunological methods, 105, 211-220, 1987, describe the use of affinity-binding using polymyxin B-Sepharose (TM) in a method for reducing endotoxin contamination in catalase and IgG solutions.
K. W. Talmadge and C. J. Siebert, in Journal of Chromatography, 476, 175-185, 1989, report separation of endotoxin from serum albumin and IgG using a polymyxin-derivatised macroporous polymer affinity column.
F. B. Anspach and O. Hilbeck, in Journal of Chromatography A, 711, 81-92, 1995, describe the use of histidine, histamine and polymyxin B affinity sorbents in separation of E. coli-derived endotoxin from serum albumin and lysozyme. The authors concluded that "an endotoxin-specific sorbent for general decontamination of protein solutions seems not to be available".
The present inventors consider that endotoxin removal from biological materials remains a problem and that there remains a need for further techniques for endotoxin removal. Accordingly, an aim of the present invention is to provide new purification procedures for the reduction of endotoxin contamination e.g. associated with recombinant nucleic acids, or with recombinant proteins, for example proteins produced in host bacteria.